Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that these compounds are not as selective as assumed and demonstrate inhibition of bombesin-stimulated phospholipase A 2 activity by both wortmannin and DMV with an IC 50 (2 nM) which is slightly more potent than the inhibition of insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate generation in these cells (ϳ10 nM). While

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... has not been possible to fully block in vitro phospholipase A 2 activity with wortmannin, inhibition cannot be a consequence of inhibition of PI 3-kinase activity since bombesin fails to generate 3-phosphorylated lipids in the intact cell. Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is not as specific as previously reported, and experimental conclusions based solely on its use should be treated with caution. The fungal metabolite wortmannin and its structural analogue demethoxyviridin (DMV) 1 have been demonstrated to have an inhibitory effect upon phosphatidylinositol 3-kinase (PI 3-kinase) activity at nanomolar concentrations (1). Inhibition has been demonstrated upon both PI 3-kinase activity in anti-p85 immunoprecipitates and the stimulation of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) generation in N-formylmethionylleucylphenylalanine (fMLP)-stimulated neutrophils (1). The binding of 17-[ 3 H]hydroxywortmannin to a number of neutrophil proteins has been demonstrated, and one of these has been identified as the 110-kDa subunit of PI 3-kinase (2). On the basis of these findings, it has been proposed that wortmannin and DMV are specific inhibitors of PI 3-kinase, and, thus, addition of these compounds to cells will result in the specific inhibition of the PI 3-kinase pathway. Consequently, an increasing number of papers have described the use of wortmannin and, on the sole basis of such experiments, assigned a role for PI 3-kinase in a number of physiological responses (e.g. . Wortmannin has also been reported to inhibit phospholipase D (PLD) (6), myosin light chain kinase, and plekstrin phosphorylation (see Ref. 1). Although these results could cast doubt upon the specificity of wortmannin, these effects have been shown to occur at concentrations greater than those reported to inhibit PI 3-kinase. However, the specificity of wortmannin for other lipid-metabolizing enzymes has not been examined. In this paper, we have examined the specificity of wortmannin and DMV and show that they inhibit stimulated PIP 2 -phospholipase C (PI-PLC), PLD, phospholipase A 2 (PLA 2 ) as well as PI 3-kinase in Swiss 3T3 cells and, in addition, in vitro PI 3-kinase and PLA 2 activities. We also demonstrate that both compounds are more potent inhibitors of PLA 2 than PI 3-kinase. . Anti-p85, clone U13 was from Serotec. SF9 cellexpressed human cytosolic PLA 2 (85-kDa enzyme) was a generous gift from Dr. C. Jackson, Fisons Pharmaceuticals, Loughborough, UK. Culture and Labeling of Cells-Swiss 3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% newborn calf serum at 37°C in a humidified atmosphere of 5% (v/v) CO 2 in air, all cells were quiesced in 2% (v/v) serum-containing medium for 24 h, with radiolabel as appropriate, prior to experiment. The cells were labeled in medium containing 2% (v/v) serum with 1 Ci/ml [ 3 H]inositol for PI-PLC experiments, 4 Ci/ml [ 3 H]palmitate for PLD experiments, and 1 Ci/ml [ 3 H]arachidonate for the PLA 2 experiments; in each case, labeling was for 24 h and the cells were grown in 24-well plates. For the measurement of 3-phosphorylated lipids, confluent cells were washed twice with phosphate-free Dulbecco's modified Eagle's medium containing 0.1% (w/v) fatty acid-free bovine serum albumin and 20 mM Hepes, pH 7.4, and subsequently labeled for 90 min in 0.25 mCi/ml [ 32 P]phosphate in the same medium. Phospholipase Assays in Intact Cells-The measurement of PI-PLC activity as the stimulated accumulation of [ 3 H]inositol phosphates in the presence of 10 mM LiCl, PLD activity as the accumulation of [ 3 H]phosphatidylbutanol in the presence of 30 mM butanol, and PLA 2 activity by the stimulated increase in [ 3 H]arachidonate generation were as described previously (7-9). PI 3-Kinase Activity-Confluent quiesced cells were washed in Hanks' buffered saline containing 10 mM glucose and 0.1% bovine serum albumin, stimulated for the required period of time, washed with ice-cold phosphate-buffered saline, and then lysed in 1% (w/v) Nonidet P-40, 10% (v/v) glycerol, 20 mM Tris-HCl (pH 8), 137 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.5 mM Na 3 VO 4 , containing 10 g/ml leupeptin and 0.2 mM phenylmethanesulfonyl fluoride. The lysates were cleared by centrifugation, and their protein concentrations were determined by the BCA method (Sigma). A solution containing 100 g of protein was incubated with 15 l of anti-p85 PI 3-kinase subunit antibody (U13 hybridoma supernatant) for 2 h at 4°C, and 20 l of 50% (v/v) Protein G-Sepharose was then added for 2 h at 4°C. The immunoprecipitates were washed successively at 4°C as follows: 2 ϫ 1 ml of lysis buffer, 2 ϫ 1 ml of 0.5 M LiCl, 0.1 M Tris-HCl, pH 8.0, 1 ϫ 1 ml of 0.1 M NaCl, 1